Multifactorial pathogenic process of allergic asthma has posed a challenge in treating this disease. IgE-mediated immediate hypersensitivity exercises two subsystems in play: (i) Upstream CD4-T-cells/IgE+ B-cells subsystem: Allergen-specific Th2/Th17 CD4 T-cells contribute to the cytokine-mediated late phase reactions. In contrast, follicular CD4 (Tfh) T-cells provide essential help to stimulate IgE+ B-cells in the germinal center (GC) and peri-GC memory B-cells (Crotty, 2011, Ann. Rev. Immunol., 29: 621). (ii) Downstream IgE network subsystem: IgE produced by the upstream Tfh/IgE B-cells initiates and amplifies a complex network of the inflammatory cell circuit of extraordinary diversity, involving a web of high affinity IgE receptor (FceRI)-bearing mast cells, basophils, eosinophils, dendritic cells and Langerhans cells, and recently neutrophils. Mezzanine intercommunication layer: Histamine released by mast cells skews dendritic cells (DCs) for Th2 preference (Lambrecht, 2009, Immunity, 31:412). FceRI on mast cells mediated IgE-dependent antigen presentation, and further augmented Th2 development (Gong, 2010, BMC Immunol, 2010, 11:34).
IgE-mediated inflammation can cause the acute phase of immediate hypersensitivity, and the late phase reaction via a plethora of IgE-produced mediators; and the IgE-FceRI cellular network can in turn enhance Th2, and the Th2-mediated late and chronic phases of allergic asthma. To add further importance to IgE is the expression of FceRI on airway smooth muscle cells for the release of TNF-a during intractable asthma. Bronchial epithelial cells also exhibit FceRI, implicated in released IL-33 and TSLP that amplify Th2-mediated inflammation (Galli and Tsai, 2012, Nat. Med., 18:693).
Besides the IgE/FceRI network, low affinity IgE receptors (FceRII, CD23) are expressed on nearly all B-cells, which mediate IgE-dependent antigen presentation for Th2 (Schmaltz, 1996, Immunol. Invest., 25: 481). FceRII on epithelial cells plays a key role in retrograde transport of IgE immune complexes in the BAL fluid, which can therefore play a role in augmenting allergen/IgE complexes-induced inflammation on intraepithelial mast cells and airway dendritic cells. The expression of FceRI on IL17AR+ neutrophils strongly suggests a new synergy of IgE and Th17-mediated inflammation in allergic asthma (Galli and Tsai, 2012, Nat. Med. 18:693; Lambrecht et al., 2009, at. Med., 31: 412).
Thus, IgE is of paramount importance in the etiology of allergic asthma by affecting IgE-mediated inflammation, a plethora of cytokines by multiple cell types, and the profound impact on Th2 (Schmaltz, 1996, Immunol. Invest., 25: 481; Gong et al., 2010, BMC Immunol., 11:34). Thus a drug candidate such as IgE B-cell vaccine targeting IgE attenuates inflammation at the multiple levels, in particular ramification of the IgE-FceRI network. Blunting IgE and IgE receptors by neutralizing anti-IgE remains the central question in treating clinical allergic asthma.
To alleviate or cure the IgE-mediated allergic diseases, it is imperative to remove circulating and mucosal IgE. In this regard, the present treatment modality focuses on the removal of circulating IgE via passive administered monoclonal antibody, Xolair. Anti-IgE, Xolair that neutralizes the receptor-binding FG loop of IgE molecules alleviates IgE-mediated allergic asthma (Chang, 2000, Nat. Biotech., 18:157). In contrast to the passive monoclonal antibody-based passive vaccine, active IgE vaccines were proposed as another treatment modality to invoke actively produced anti-IgE that neutralizes host's IgE. One approach resides in random chemical coupling of synthetic IgE peptides to the immunogenic protein carriers as active vaccines (Brown et al., 2009, U.S. patent application Ser. No. 12/634,336).
Another embodiment of invention resides in engineering neutralizing IgE B-cell epitopes within thermostable, immunogenic protein scaffold in a single step internally (Chen, 2008, U.S. patent application Ser. No. 12/011,303; Chen, 2008, J. Immunol. Meth., 333: 10). The present embodiment of the invention represents constraining native IgE B-cell epitopes in two internal steps: into super b-strands, and further into the cystine knots; and then integrated in one external step onto the protein scaffold, of which the thermostability of the immunogenic protein scaffold is not compromised by foreign loop insertion.
Conception of a monospecific B-cell epitope and its conjugation as synthetic peptide unto an immunogenic protein was pioneered by Atassi, Lerner and Brown in the late 80's (Rowlands et al., 1983, Nature, 306: 694; Atassi, 1978, Immunochem., 15: 909). Most antigenic structure are presented as a loop constrained by the secondary alpha helix and beta sheet structure, and properly folded in the three dimensional array determined by favorable energetics.
Through extensive studies of numerous potential B-cell candidate epitopes, a B-cell loop antigenic epitope, taken out from the native constrained secondary and tertiary protein folding, is distorted in conformation. Such synthetic or recombinant peptides randomly conjugated to or integrated to a protein carrier backbone exhibited thermodynamically unpredictable, multiple distorted, random conformations (Rowlands, et al., 1983, Nature, 306:694). Synthetic or recombinant linear IgE B-cell epitopes without proper constraint remain in a state of complex random array without definable structural integrity. Constraining scaffold in supporting the antigenic loop is required for enabling functional native conformation with structural integrity.
In contrast, conception of constrained IgE B-cell epitopes prompts the step to constrain the IgE B-cell epitope directly in the thermostable protein scaffold, whereby functional native conformation of the constrained neutralizing IgE B-cell epitopes can be enabled by the constraint. The embodiment of this invention further improves the constraining platform in placing B-cell epitopes into the super constraining beta (b)-strands, and further strengthened by the thermostable cystine knots, and finally integrated onto another thermostable protein scaffold, engineered in an optimal oxidative folding chemical milieu. Hence the embodiment of the three improvements in this invention enables the native expression and structural integrity of the B-cell epitopes.
The embodiment of this invention with active, conformationally constrained IgE B-cell epitope vaccine improves over the passive neutralizing anti-IgE monoclonal antibody (Chang, 1995, U.S. Pat. No. 5,428,133): (i) Sustained circulating IgE-Xolair complexes in treated patients cause long-term IgE suppression. The regimen requires 36 to 54 week-long treatment in order to neutralize 95% circulating IgE. However, due to the small size of immune complexes, circulating IgE/IgG1 Xolair complexes assume a half-life of 21 days of IgG1 (IgE lasts only one day); consequently, total circulating IgE in the complexes are persistently elevated ˜100 fold as a result of treatment (Chang, 2010, Nat. Biotech., 18:157).
Active IgE B-cell vaccines embodied by this invention improve the safety margin by producing active polyclonal anti-IgE in the vaccinated recipients with appropriate length of protection based on the vaccination/booster regimen. The duration is controlled by reactivation of memory CD4 helper T-cells to the protein scaffold. Due to the polyclonal antibodies, the clearance of circulating and mucosal IgE and IgG complexes will be efficient via the liver sinusoids and Kupffer cells. Furthermore, as a murine human chimera antibodies, Xolair causes anaphylaxis in individuals (3.14/1000 patients vs 5.4 events/million shots), the constrained IgE B-cell active vaccine induces endogenous autologous anti-IgE indigenous to the host.
(ii) Xolair is inefficient in targeting the mission-critical pathogenic IgE in the lung. The passively delivered Xolair via the subcut route, sieved through afferent lymphatics into thoracic duct lymph into the general blood circulation, without permeating into the critical sites of the lung, central for allergic asthma. Allergen-specific IgE, secreted by IgE plasma cells present in induced peribronchus-associated lymphoid tissues (iBALT), into the surrounding lamina propria under the bronchial epithelial and endothelial cells, remains inaccessible to circulating anti-IgE monoclonal antibodies, whose delivery depends solely on inflammation-mediated changes in vascular permeability (Lambrecht, 2009, Nat. Med., 31: 412).
A further embodiment of this invention is that IgE B-cell vaccines delivered via mucosa route of immunization elicit anti-IgE in iBALT that neutralizes pathogenic IgE in situ in the iBALT. The FG super b-strands constrained IgE B-cell epitopes with or without Min-23 cystine knot constraint, integrated onto the immunogenic protein scaffold can be employed as IgE B-cell vaccines. B-cells recognizing native, constrained FceRIa receptor-binding IgE-B-cell epitopes are activated by CD4 helper T-cells reactive with CD4 helper epitopes on the immunogenic protein scaffold. Anti-IgE antibodies of the IgA and IgG classes can be released directly in mucosal secretion in addition to circulation via a preferred mucosal route of immunization with FDA-approved adjuvants, Toll like receptor (TLR)-7 agonist imiquimod, alum, lipid A-based adjuvant or TLR-9 adjuvant presently being evaluated.
The designed constrained IgE B-cell vaccines elicit polyclonal neutralizing anti-IgE of the IgG and IgA classes that inhibits IgE-mediated mast cell degranulation, and prevents airway inflammation and airway hyper-reactivity (Ahr) (Zuberi et al, 2000, J. I., 164: 2667). Thus engineering constrained IgE B-cell epitopes in the FG super b-strands and cystine knots, integrated onto the immunogenic protein scaffold yields can lead to new anti-IgE pan-allergy vaccines that can benefit asthmatics of different disease spectra through mucosal IgE targeting and neutralization.